Peptide libraries have emerged as a powerful resource to identify therapeutically relevant molecules. In addition, peptide libraries are also relevant resources for many other purposes and for basic research.
Therapeutically, peptides have certain advantages over small molecules and large biologics, such as antibodies. As compared to small molecules, peptides typically have a larger interaction interface with an antigen, which comprises hydrogen bonds and van der Waals forces. This leads to high affinity binding, a high specificity for the antigen and typically a high potency. As compared to antibodies, peptides are much smaller and therefore typically penetrate tissue more easily. Certain tumors are inaccessible for antibody therapy.
Numerous phage display peptide libraries do exist, including libraries that utilize constrained peptides. Constraint peptides overcome certain disadvantages that are associated with linear peptides, including weak binding affinities due to a higher conformational flexibility, and an increased susceptibility to proteolytic degradation in the human body.
A natural occurring constrained motif of (poly)peptides is the α-helical bundle. α-helices constitute the largest class of protein secondary structures and play a major role in mediating protein-protein interactions. However, short synthetic peptides of 10-30 amino acids in length are usually not thermodynamically stable helices in water and adopt only random structures (Harrison et al., Proc. Natl. Acad. Sci. USA. 2010 Jun. 29; 107(26)).
α-helical bundles can appear in different forms, including two, four, or even multiple bundles. The individual α-helical peptides in such bundle proteins may be orientated in a parallel or anti-parallel arrangement, thus forming coiled-coil structures in which the helical axes are aligned slightly offset from one another.
The α-helical structures that occur in such bundles usually comprise heptad repeats with a profile consisting of a hydrophilic exterior, a hydrophobic interior and a border of polar amino acid residues that form interhelical salt bridging interactions.
Many natural occurring proteins, like keratin, myosin, epidermin, fibrinogen and tropomysin, have a coiled-coil structure formed by two dimerized α-helical peptides. Furthermore, coiled-coil structures are frequently found on DNA binding proteins, where this motif is referred to as a leucine zipper.
Coiled-coil domains are also found in the Jun, Fos (O'Shea et al., Science. 245:646-648 (1989)), C/EBP (Landschultz et al., Science. 240: 1759-1764 (1988)) and for instance in GCN4 binding proteins (O'Shea et al., Science. 242:538-542 (1989)). Naturally occurring α-helical coiled-coil structure are often found in a parallel orientation, which is thought to be a stable conformation.
Approaches have been described to adapt such structures to design specific recognition molecules. WO94/29332 describes polypeptides containing anti-parallel coiled-coils wherein said scaffolds were modified to incorporate helical recognition sequences from naturally-occurring proteins such as DNA binding proteins and cytokines.
U.S. Pat. No. 5,824,483 describes the construction of a de novo designed and chemically synthesized combinatorial library of α-helical peptides. The α-helical peptides were stabilized by intrahelical lactam bridges and optionally by an additional second α-helical peptide thus resulting in a dimeric coiled-coil structure in a parallel or anti-parallel orientation. However, the only enabled peptide library encompassed a single 24 amino acid long α-helical polypeptide chain which is stabilized via two intrahelical lactam bridges and which is diversified at 5 amino acid positions.
Fujii et al. (Tetrahedron Letters 42, 3323-3325 (2001)) describes a more specific approach for a helix-turn-helix based library. The scientific publication discloses a de novo chemically synthesized anti-parallel orientated helix-turn-helix peptide library wherein the amino- and carboxyl-terminal peptides are linked via a glycine based linker. Each of the two helix-turn-helix forming peptides consisted of 14 amino acids and was stabilized by hydrophobic interactions with leucine residues on the two respective helices. In contrast to the library of the present disclosure, only the carboxyl-terminal helix peptide was diversified at 3 solvent exposed positions with a mixture of 5 naturally occurring amino acid residues.
A complementary method for utilizing a peptide library is the display of such libraries on filamentous bacteriophages. This method allows the preparation of libraries as large as 1010 unique peptide members, many orders of magnitude larger than libraries that may be prepared synthetically.
A phage displayed anti-parallel orientated helix-turn-helix peptide library was described by Fujii and Coworkers in 2008 (Biochemistry, 47, 6745-6751 (2008)). In contrast to the above mentioned library, the carboxyl-terminal helix peptide was randomized at 5 solvent exposed regions yielding in a theoretical library size of 3.2×106. The library was displayed on the major coat protein VIII of filamentous phage with a glycine/serine linker in conjunction with a detectable tag. A particular utilization of the helix-turn-helix peptide library to generate “Microantibodies” has been further described by Fujii et al. in 2011 (Drug Delivery System, 26-6, 2011, p. 593-603), in 2009 (Yakugaku Zasshi, 129 (11), 1303-1309, 2009) and 2013 (Current Protocols in Chemical Biology, vol. 5 (3), 171-194, 2013)
A common structural feature of the two libraries described by Fujii and Coworkers is the predominant usage of alanine at solvent exposed positions of the two α-helical peptides. Stretches of alanine (poly alanine) are known to facilitate formation of α-helical structures but they also may display low solubility in aqueous solutions and thus are prone for aggregation.
More importantly, the libraries described by Fujii and Coworkers are only diversified within the carboxyl-terminal α-helical peptide by diversifying solvent exposed alanine positions. In this scenario, the non-diversified amino-terminal peptide is thought to retain its α-helical structure and to stabilize the helix-turn-helix structure of the molecule. However, randomization of the carboxyl-terminal α-helical peptide as provided by Fujii still resulted in library members with undesired multiple random like conformations which required a particular purification process in order to enrich for correctly folded helix-turn-helix structures (Fujii et al. (Tetrahedron Letters 42, 3323-3325 (2001)).
A major disadvantage of diversifying only one of the two α-helical peptides lies in the fact that the approach significantly limits the achievable library size and significantly narrows down the interaction interphase between the polypeptides of the library and their bound target molecules of interest resulting in reduced specific and affinity.
Based on limitations of the above mentioned approaches, there is still an unmet need to develop improved helix-turn-helix polypeptide libraries of considerable size.
The library of the present disclosure differs in multiple ways from the libraries disclosed by Fujii. The design of the library of the present disclosure is based on a combined approach taking into account statistical, structural and rational factors. This included in a first instance the analysis and use of the most abundant amino acid residues found at given positions in natural occurring α-helical structures. Such amino acids are considered to have favorable biophysical properties including low immunogenicity, resistance against temperature and chemical denaturation, relative insensitiveness for pH alterations, serum stability and resistance against proteolytic degradation by proteases.
Secondly, the variable positions within the helix-turn-helix library of the present disclosure are present on both, the amino- and the carboxyl terminal α-helical peptide and are displayed in the same relative parallel orientation. This two features enable the formation of a wide and flat interaction interface over the whole length of the helix-turn-helix molecule. The enlarged interaction interface is crucial for an optimal protein-protein interaction with a target antigen of interest resulting in improved specificity and affinity, both critical aspect in the development of therapeutic molecules.
Furthermore, in order to prevent a potential destabilization of the helix-turn-helix scaffold caused by the introduction of a large number of variable positions in both α-helical peptides, additional structural consideration for promoting helix formation and stabilizing the helix-turn-helix structure were taken into account to select the most appropriate amino acid residue at each invariant position. These amino acid residues were selected to stabilize the molecular structure by inter- and intrahelical electrostatical interactions and interhelical hydrogen bonding.
In summary, the library of the present disclosure overcomes the limitations of the helix-turn-helix libraries disclosed by Fujii and Coworkers by maximizing the number of diversified positions without compromising the stabilizing α-helical structures leading to a more efficient development of the resulting polypeptides and an increase in safety and efficacy of the respective therapeutics in patients.